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1/10/2014 EURL Campy 1 DTU Food, Technical University of Denmark Monitoring of antimicrobial resistance in Campylobacter – EURL AR activities in framework of the new EU regulation Lina Cavaco [email protected]

Monitoring of antimicrobial resistance in Campylobacter ...€¦ · Monitoring of antimicrobial resistance in Campylobacter – EURL AR ... in the antimicrobial susceptibility testing

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Page 1: Monitoring of antimicrobial resistance in Campylobacter ...€¦ · Monitoring of antimicrobial resistance in Campylobacter – EURL AR ... in the antimicrobial susceptibility testing

1/10/2014EURL Campy1 DTU Food, Technical University of Denmark

Monitoring of antimicrobial resistance in Campylobacter – EURL AR activities in framework of the new EU regulation

Lina Cavaco [email protected]

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1/10/2014EURL Campy2 DTU Food, Technical University of Denmark

Outline• EURL-AR presentation and tasks• Tasks related to new monitoring• Brief overview Campylobacter in the monitoring

– Sampling – AST and panel of antimicrobials to be tested

• Ring trials for Campylobacter AST in EURL-AR network• Results of most recent Campylobacter AST trial (2013)• Challenges

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1/10/2014EURL Campy3 DTU Food, Technical University of Denmark

The tasks of the EURL-AR • Scientific advice and support to the Commission

• Co-ordination of National Reference Laboratories and provision of technical support

– Creation of the network of NRL’s– Workshops – Dissemination of knowledge and information– Collection of information on activities at the NRL’s

• Ring trials, comparative testing and quality assurance

• Confirmatory testing

• Evaluation and development of analytic methods

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Co-ordination of National Reference Laboratories and provision of technical support

– Creation of the network of NRL’s• The address list

– Workshops• One annual workshop next April 2015

– Training courses aimed at the specific methods to apply in the monitoring (2013 was training on AST incl. Campylobacter , this year was focused on selective isolation of presumptive ESBL-, AmpC or carbapenemase producing E. coli

– Site visits– Dissemination of knowledge and information

• Home page: www.eurl-ar.eu• Newsletters• E-learning site ( ongoing preparation)

– Collection of information on activities at the NRL’s• Questionnaires

The Tasks of the EURL-AR

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1/10/2014EURL Campy5 DTU Food, Technical University of Denmark

The tasks of the EURL-AR (The ring trial system - EQAS)

• AST of eight Salmonella, Campylobacter, E.coli, Staphylococcus andEnterococcus strains

• MRSA isolation identification and spa typing• Genotyping (gene EQAS)• New one on the way, awaiting approval of WP 2015• Supply labs with original ref. strains E. coli ATCC 25922, C jejuni ATCC

33560, S. aureus ATCC 29213 and E. faecalis ATCC 29212

• Results and comments submitted through a web-based data entry program, using individual passwords

• Instant individual evaluation reports with suggestions for corrective actions

• Yearly over-all evaluation reports on the web

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1/10/2014EURL Campy6 DTU Food, Technical University of Denmark

Work of EURL-AR and the monitoring according to EU 652/2013

• Participation in the EFSA Workgroups• Scientific advice to the EU Commission and participation on

the Preparation and discussion of the contents and methods used in the regulation

• Facilitating training• Facilitating ring trials• Development of methods and confirmatory testing

Thus, the activities aim at implementing, from an analytical point of view, the provisions of monitoring of antimicrobial resistance set down the EU regulation 652/2013

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Campylobacter monitoring acc EC 652/2013• Sampling

– (a) In the years 2014, 2016, 2018 and 2020 for laying hens, broilers and fresh meat thereof, and fattening turkeys.

– (b) In the years 2015, 2017 and 2019, for pigs, bovines under one year of age, pig meat and bovine meat.

• Sample nrs– 170 isolates for AST for each combination of bacterial species and type of

sample of animal population or food category listed in point 1(a), (b), (c), (e) and (f). in Member States with a production of less than 100 000 tonnes -85 isolates is minimum

– In Member States with low bacterial prevalence or low number of epidemiological units, all available isolates at the end of the monitoring period shall be included in the antimicrobial susceptibility testing.

Disclaimer: for exact information and further info on sampling design and representative sampling - consult the regulation and the EFSA technicalspecifications for the sampling procedures!

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Campylobacter monitoring• Testing

– ID Campylobacter C. jejuni /C. coli• Recommended PCR method for speciation under:

http://eurl-ar.eu/data/images/protocols/protocol%20for%20campylobacter_november_2013.pdf

Acc: Denis, C. Soumet, K. Rivoal, G. Ermel, D. Blivet, G. Salvat and P. Colin. Development of a m-PCR assay for simultaneous identification of Campylobacter jejuniand C. coli. Letters in Applied Microbiology 1999, 29, 406–410

– AST • MIC testing• Antimicrobial panel and ranges acc EFSA technical

specifications not changed from previous reccomendations– QC and storage of isolates (for 5 years)

• Data collection- isolate-based data - EFSA

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1/10/2014EURL Campy10 DTU Food, Technical University of Denmark

Choice of antimicrobials and ranges• Insert reasons from EFSA

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1/10/2014EURL Campy12 DTU Food, Technical University of Denmark

Rationale for antimicrobials to be tested• Macrolides (erythromycin, clarithromycin, azithromycin) are considered

first choice drugs for therapy of human infections with Campylobacterspp. Acquired resistance is caused by single point mutations in the ribosomal DNA resulting in cross-resistance between the different macrolides. Macrolides are commonly used in food-animals and resistance to macrolides is reported, predominantly in C. coli. Erythromycin is commonly used in the test panels as representative of the macrolides.

• (Fluoro)quinolones (ciprofloxacin, nalidixic acid) Resistance to quinolones is due to mutation in the gyrase gene resulting in resistance.

Both nalidixic acid and ciprofloxacin are used in existing monitoringprogrammes as indicators of acquired resistance to quinolones in Campylobacter spp.

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1/10/2014EURL Campy13 DTU Food, Technical University of Denmark

Rationale for antimicrobials to be tested(cont)• Tetracyclines are considered alternative choices for treatment of

Campylobacteriosis. But acquired resistance does occur. Therefore inclusion of this class is relevant. Resistance in Campylobacter spp. is encoded by tet(O), a gene that expresses a ribosomal protection protein.

• Streptomycin Resistance to aminoglycosides in Campylobacter spp. is encoded by APH and AAD-genes.

Although cross resistance between different aminoglycosides is incomplete, streptomycin resistance can be used as indicator for acquired genes.

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1/10/2014EURL Campy14 DTU Food, Technical University of Denmark

Ring trials, comparative testing and quality assurance

• Ring trials, comparative testing and quality assurance– Proficiency tests -include one annual ring trial on AST of

Campylobacter

• Confirmatory testing– Confirmation of results and characterization of monitoring

isolates

• Evaluation and development of analytic methods– Establishing reference strain collections for relevant research

and requests– Methods for detection of relevant resistances- very focused on

the needs in monitoring.

The tasks of the EURL-AR

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•Area: Antimicrobial Resistance•Microorganisms:

–Campylobacter–Salmonella–E. coli–Enterococci –Staphylococci–(MRSA, Genotyping and ESBL/AmpC)

•Methods for phenotypic antimicrobial susceptibility testing (AST): –Microbroth / Agar dilution (MIC-test)

Focus of the EURL-AR EQAS’s

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•To have laboratories evaluate their performance of antimicrobial susceptibility testing (AST)

•To assess the quality of AST in European reference laboratories

•And improve the quality of monitoring data to EFSA

•To identify challenges within the methods•To evaluate the effect of training courses

Objectives of the EQAS

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•Accredited methods (relevant for the EQAS): – ‘MIC testing’ according to EN/ISO 17025– ‘Provider of EQAS (serotyping and AR)’ according to ILAC G13

(International Laboratory Accreditation Cooperation)

•International standards/guidelines– ISO (International Organization for Standardization)– CLSI (Clinical and Laboratory Standards Institute) – EUCAST (The European Committee on Antimicrobial Susceptibility

Testing – www.eucast.org)

Quality Assurance of the EQAS

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Campy EQAS AST- Participation 2013

28 countries

30 sets of Campylobacterresults

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Comparison to former EQASs

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Campylobacter results – strains

Test strain AST in total

% correct

C-8.1 (C. coli) 179 93.9C-8.2 (C. coli) 180 97.8C-8.3 (C. jejuni) 174 94.8C-8.4 (C. jejuni) 161 96.9C-8.5 (C. jejuni) 180 97.8C-8.6 (C. coli) 180 96.1C-8.7 (C. coli) 180 96.1C-8.8 (C. coli) 180 98.9

Only 3 incorrect identifications out of 240 identifications reported > 98.8 % correct

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Campylobacter results – antimicrobials

Antimicrobial % of correct ASTCiprofloxacin 96.6Erythromycin 97.5Gentamicin 99.6Nalidixic acid 93.2Streptomycin 96.6Tetracycline 95.7

Overall percentage AST correct: 96.5%

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1/10/2014EURL Campy22 DTU Food, Technical University of Denmark

Most frequent problems

• Campylobacter– Standardization and harmonization of methods– Sometimes growth issues on MIC plates (EURL-Campy

contacted a few times)– Issues with acquisition or supplementation of media– Less problems with AST results now that disk diffusion results

are not accepted (formerly some labs encountered problems due to the method used

– New challenges with new monitoring not yet observed

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1/10/2014EURL Campy23 DTU Food, Technical University of Denmark

Thank you!!

From the EURL-AR team and our network